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DJ-1 modulates the expression of CuZn-superoxide dismutase-1 through the Erk12ЦElk1 pathway in neuroprotection.

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ORIGINAL ARTICLE
DJ-1 Modulates the Expression of Cu/ZnSuperoxide Dismutase-1 through the
Erk1/2–Elk1 Pathway in Neuroprotection
Zhiquan Wang, PhD,1,2,3 Jun Liu, MD, PhD,2 Siyan Chen, MS,2 Ying Wang, MS,2
Li Cao, MD, PhD,2 Yu Zhang, PhD,2 Wenyan Kang, MS,2 Hui Li, MS,2 Yaxing Gui, PhD,2
Shengdi Chen, MD, PhD,1,2 and Jianqing Ding, MD, PhD1,2
Objective: Loss of function mutations of Park7/DJ-1 gene increase the susceptibility of dopaminergic cells to
reactive oxygen species and cause early onset familial Parkinson disease (PD). However, the mechanisms underlying
dopaminergic neuron loss related to DJ-1 mutation remain undefined. Therefore, it is important to find the new
mechanisms underlying the antioxidative functions of DJ-1.
Methods: DJ-1 knockdown cells and DJ-1 knockout mice were used to elucidate the mechanisms underlying the
antioxidative stress of DJ-1. Preliminary study of the saliva from PD patients and controls was used to confirm our
findings obtained from the above studies.
Results: Our experiments showed that DJ-1 interacted with Erk1/2 and was required for the nuclear translocation of
Erk1/2 upon oxidative stimulation. The translocation of Erk1/2 activated Elk1 and sequentially promoted superoxide
dismutase1 (SOD1) expression. The nuclear translocation of Erk1/2, the activation of Elk1, and the ensuing
upregulation of SOD1 were all suppressed in DJ-1 knockdown cells and DJ-1 null mice treated with oxidative insult.
Furthermore, reintroduction of SOD1 into DJ-1 knockdown cells protected them against oxidative stress. Finally, in
the preliminary study, we found close correlation between the protein levels of DJ-1 and SOD1 in the saliva samples
from different stages of PD patients.
Interpretation: Our studies suggest that DJ-1 regulates SOD1 expression through Erk1/2–Elk1 pathway in its
protective response to oxidative insult.
ANN NEUROL 2011;70:591–600
P
arkinson disease (PD) is a common neurodegenerative
disease that affects approximately 1% among persons
65 years of age and 5% among those 85 years or older.1
The featured pathological change of PD is the selective
and progressive loss of dopaminergic (DA) neurons, but
the molecular mechanism underlying neuronal death still
has not been clarified.2 Nevertheless, increasing evidence
has suggested that both environmental and genetic factors
play important roles in the pathogenesis of the disease.3
Epidemiologic studies have suggested that increased
PD is associated with overexposure to environmental factors such as iron, manganese, herbicides, and pesticides.4,5 These neurotoxins are believed to induce oxida-
tive stress, which is thought to be a significant cause of
PD.6,7 Studies in hereditary PD revealed that genetic factors also played an important role in the development of
PD. More than 13 loci and 9 genes have been identified
to date.8 Among these genes, DJ-1 has attracted much
attention recently because of its association with both
autosomal recessive early onset PD and sporadic PD.9
Several PD-causing mutations have been identified,
including exon deletions, truncations, and homozygous
and heterozygous point mutations, which are all predominantly characterized by loss of function.10
DJ-1 protein belongs to the ThiJ/PfpI superfamily
and is expressed in both neurons and astrocytes.11,12 The
View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana.22514
Received Jul 20, 2010, and in revised form May 23, 2011. Accepted for publication Jun 2, 2011.
Address correspondence to Dr Ding, Institute of Neurology, Ruijin Hospital, 197 Ruijin Er Road, Shanghai 200025 P.R. China. E-mail: jqding18@yahoo.
com or Dr Chen, Department of Neurology, Ruijin Hospital, 197 Ruijin Er Road, Shanghai, P.R. China; E-mail: chen_sd@medmail.com.cn
From the 1Laboratory of Neurodegenerative Diseases, Institute of Health Science, Shanghai Institutes for Biological Sciences, Chinese Academy of Science
and Shanghai Jiao Tong University School of Medicine; 2Department of Neurology and Institute of Neurology, Ruijin Hospital, Shanghai Jiao Tong
University School of Medicine and; 3Graduate School of the Chinese Academy of Science Shanghai, China.
Additional supporting information can be found in the online version of this article.
C 2011 American Neurological Association
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exact biological function of DJ-1 is still unclear; however,
increasing evidence has implied that DJ-1 responds to
oxidative insult and might behave as a hydroperoxideresponsive protein.13,14 DJ-1 expression is induced in
cells that have been subjected to oxidative stress, and its
function is to protect cells against oxidative insult.13,14
Enhancement of DJ-1 level has also been found in the
cerebrospinal fluid of sporadic PD patients.15 Moreover,
postmortem studies of brain samples from sporadic PD
patients have shown abnormal oxidized forms of DJ1.9,12 These all strongly indicate that DJ-1 may play an
important role in the pathogenesis not only in inherited
PD but also in sporadic PD. The above hypothesis is
further supported by the observation that knockdown
(KD) or knockout (KO) DJ-1 increased cell death by
rendering cells more susceptible to oxidative stress.14,16
DJ-1–deficient mice have no obvious dopaminergic neuron loss but are hypersensitive to 1-methyl-4-phenyl-1, 2,
3, 6-tetrahydropyrindine (MPTP) and oxidative
stress.17,18 DJ-1 is an atypical peroxiredoxinlike peroxidase that scavenges peroxides through oxidation of Cys106.19 Numerous studies have demonstrated that DJ-1
plays a part in the cellular defensive response to oxidative
stress, but the molecular mechanism of how mutations of
DJ-1 result in PD is still a puzzle.
Because loss of function mutations in the DJ-1
gene are correlated with the degeneration of DA neurons
and autosomal recessive PD,20 in this report we
employed DJ-1 KD cells and DJ-1 KO mice to study the
antioxidative defense mechanism of DJ-1. Oxidative stress
is a state of oxidative damage in the cell, tissue, or organ
caused by the reactive oxygen species (ROS). Superoxide
anion (O
2 ) is among the major ROS and can be converted into hydrogen peroxide (H2O2) by superoxide dismutases (SODs).21,22 The toxic H2O2 is broken down
into nontoxic water and oxygen by peroxidase. Our
experiments have shown that DJ-1 regulated SOD1
expression through the Erk1/2–Elk1 pathway to protect
cells against oxidative insult. KD or deletion of DJ-1
diminished its regulation of the expression of SOD1 upon
oxidative stimulation and enhanced cell death.
Materials and Methods
Reactive Oxygen Species Measurement
Intracellular ROS production was measured by 5-(and-6)-chloromethyl-20 , 70 -dichlorodihydrofluorescein diacetate assay.23
Both general (mitochondrial plus cytosolic) and specific (mitochondrial or cytosolic) hydrogen peroxide levels were measured
by Amplex Red regents and horseradish peroxidase. General
(mitochondrial plus cytosolic) superoxide level was measured by
dihydroethidium (DHE) as previous described.24 Protocol of
Dr Melov’s laboratory25 was modified to detect the specific (mi-
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tochondrial or cytosolic) superoxide level. Briefly, 4lg of mitochondria or 10lg of cytosol fraction was incubated with 5lM
DHE in a 96-well plate at 37 C for 60 minutes. Ten micrograms of sonicated salmon DNA and 0.6% Triton were then
added into the wells. The fluorescence was measured by excitation at 544nm and emission at 612nm with a Microplate
reader (Safire 2C; Tecan, Männedorf, Switzerland). The mitochondria and cytosol fractions were separated by the Mitochondria Isolation Kit for Cultured Cells (89874; Pierce Biotechnology, Rockford, IL) and were used to measure ROS (superoxide
or hydrogen peroxide) separately.
The rest of the materials and methods are described in
the Supplementary Materials.
Results
ROS Production and Cell Death Were Increased
in DJ-1–Deficient Cells
To explore the mechanisms underlying the loss of function
mutation of DJ-1, vector-based shRNA was used to establish DJ-1 KD SH-SY5Y stable lines. Western blot examination showed that DJ-1 was silenced efficiently (Fig 1A).
DJ-1 KD cells treated with 1-methyl-4-phenylpyridinium
(MPPþ) and paraquat produced more ROS and were
more vulnerable to oxidative stress than scramble SHSY5Y cells (see Fig 1B, C). ROS mainly include superoxide anion and hydrogen peroxide, and MPPþ can induce
the formation of superoxide anion as well as hydrogen
peroxide. Therefore, the superoxide and hydrogen peroxide levels were also measured. The results showed that
DJ-1 KD could sensitize the cells to produce more superoxide and hydrogen peroxide (see Fig 1D, E). These
results suggested that DJ-1 KD cell lines were more susceptible to MPPþ or paraquat-induced cell death as a
consequence of the excessive production of ROS.
DJ-1 Deficiency Suppresses SOD1 Expression,
and Reintroduction of SOD1 into DJ-1–Deficient
Cells Can Decrease the ROS Production
SOD is among the major antioxidative factors in the
nervous system. The wild-type (WT) and DJ-1 null
mouse were administered MPTP to explore whether
SODs participate in the process of the DJ-1 deficiencyinduced neuron loss upon MPTP insult. The peak of
striatal MPPþ level is often found approximately 90
minutes after MPTP administration.26 As expected, the
expression of SOD1 was increased at 60 and 90 minutes
after MPTP administration and was most remarkable at
60 minutes; however, the upregulation of SOD1 was
abolished in the DJ-1 KO mice. SOD2 was slightly
enhanced in both WT and DJ-1 KO mice treated with
MPTP (Fig 2A and supplementary Fig. S1 A-B). The
upregulation of SOD1 expression was also observed in
SH-SY5Y cells treated with MPPþ or paraquat, but this
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FIGURE 1: Knockdown DJ-1 in SH-SY5Y enhanced reactive oxygen species (ROS) production and cell death when cells were
attacked by 1-methyl-4-phenylpyridinium (MPP1) or paraquat. (A) Western blot result showed that DJ-1 was efficiently silenced
in the DJ-1 shRNA SH-SY5Y cells compared to scramble cells. (B) DJ-1–deficient SH-SY5Y cells were treated with MPP1 or paraquat. 5-(and-6)-chloromethyl-20 , 70 -dichlorodihydrofluorescein diacetate assay was used to check the ROS production. The
results showed that there was significant difference between DJ-1 knockdown SH-SY5Y cells and control cells. *p 5 0.017. (C)
WST-1 assay showed that DJ-1–deficient SH-SY5Y cells are more susceptible to MPP1 or paraquat insults. **p 5 0.0001. (D)
DJ-1–deficient SH-SY5Y cells were treated with MPP1. Amplex Red assay was used to check the hydrogen peroxides levels.
The results showed that there was significant increase of hydrogen peroxides in the DJ-1 knockdown SH-SY5Y cells compared
to the control cells. *p < 0.05. (E) DJ-1–deficient SH-SY5Y cells were treated with MPP1. Dihydroethidium (DHE) assay was
used to check the superoxide production. The results showed that there was significant increase of superoxide in the DJ-1
knockdown SH-SY5Y cells compared to the control cells. *p < 0.01.
upregulation was inhibited in the DJ-1 KD cells (Fig 2B
and supplementary Fig S2 A-B). Furthermore, ROS production was decreased after SOD1 was reintroduced into
DJ-1 KD SH-SY5Y cells (Fig 2F). These observations
indicated that the antioxidative effect of DJ-1 may be
accomplished through the regulation of SOD1 expression.
SOD1 has been found mainly in cytosol, whereas SOD2
has been found mainly in the mitochondria.27 To further
confirm the effect of DJ-1 deficiency on SOD1 expression
and ROS production, the superoxide levels of cytosol and
mitochondria were examined in DJ-1 KD SH-SY5Y and
control cells. It showed that MPPþ-induced cytosolic
superoxide was increased. However, there was no significant change of mitochondria superoxide (Fig 2D and E).
We also measured the hydrogen peroxide level in DJ-1 KD
SH-SY5Y and control cells. Consistent with the previous
report that DJ-1 is an atypical peroxiredoxinlike peroxidase,19 the mitochondria peroxide production was intensified when DJ-1 was knocked down. MPPþ-induced cytosolic peroxide levels were not changed in DJ-1 KD cells
(Supplementary Fig S3 A-B).
Elk1 Could Bind to SOD1 Promoter, and
Inhibition of Elk1 Suppressed the ROS-Induced
SOD1 Transcription Activation
Previous studies have reported that the transcription factor
Elk1 can bind to rat SOD1 promoter and increase SOD1
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gene transcription.28 Using the online tool Genomatix, we
found that there are 2 potential Elk1 binding sites in
SOD1 promoter. ChIP (chromatin immunoprecipitation)
assay confirmed the binding of Elk1 and SOD1 promoter
(Fig 3A and B). Phosphorylation of Elk1 is thought to enable Elk1 to mediate transcriptional activation. Phosphorylated Elk1 and SOD1 expression were increased upon the
MPPþ or paraquat insult in WT SH-SY5Y cells. To elucidate the functional relationship between Elk1 and SOD1,
the phosphorylation site of Elk1 was mutated to generate a
dominant negative form of Elk1 (S383A).29 SH-SY5Y cells
were transfected with Elk1 S383A construct and treated
with MPPþ or paraquat. The phosphorylation of Elk1
resulting from MPPþ or paraquat treatment was inhibited
(Fig 3C and Supplementary Fig S4 A). Luciferase assay
showed that Elk1 S383A inhibited the activation of SOD1
promoter induced by MPPþ and paraquat (Fig 3E). Correspondingly, the SOD1 expression was decreased, and ROS
production was enhanced in Elk1 S383A-transfected cells
(Fig 3C and supplementary Fig S4 A and C). These demonstrated that the activation of SOD1 promoter by MPPþ or
paraquat is controlled by transcription factor Elk1.
SOD1 Expression is Erk1/2-Elk1 MAPK Pathway
Dependent
Elk1 is phosphorylated by upstream MAPK kinases
including Erk1/2. To explore whether Erk1/2 participated
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FIGURE 2: DJ-1 exerts its protective function against oxidative stress through SOD1 expression. (A) Wild-type (WT) and DJ-1
knockout (KO) mice were administered 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyrindine (MPTP) for 60 and 90 minutes. SOD1
and SOD2 expression levels in the substantia nigra were examined by Western blot. (B)SH-SY5Y cells were treated with 1methyl-4-phenylpyridinium (MPP1). SOD1 protein level was measured by Western blot. (C) The upregulation of SOD1 upon
MPP1 treatment was suppressed in the DJ-1 knockdown SH-SY5Y cells. (D) The cytosolic superoxide level of the DJ-1 knockdown SH-SY5Y cells and scramble cells was measured. DHE 5 dihydroethidium (E) The mitochondria superoxide level of the
DJ-1 knockdown SH-SY5Y cells and scramble cells was measured. NS 5 not significant. (F) SOD1 was reintroduced into the DJ1 knockdown SH-SY5Y cells; reactive oxygen species (ROS) production upon MPP1 and paraquat treatment was compared
between SOD1 transfected cells and blank vector transfected cells. *p 5 0.01, **p 5 0.037.
in the regulation of SOD1 expression, the effects of Erk1/
2 on the expression of SOD1 were examined. Phosphorylation of Erk1/2 was increased in MPPþ-treated SH-SY5Y
cells (Supplementary Fig S5A). Pretreatment of MEK-Erk
inhibitor PD98059 could suppress the upregulation of
SOD1 induced by MPPþ (Fig 4A), whereas CA-MEK1
(constitutively active MEK1, the upstream activator of
ERK1/2)30 could increase the SOD1 expression in a dosedependent manner (see Fig 4B). The activation of SOD1
promoter was suppressed (Fig 4C), and ROS production
was enhanced by PD98059 pretreatment when cells were
exposed to MPPþ or paraquat (Supplementary Fig S5B).
Furthermore, the pretreatment of PD98059 could intensify primary neuronal death induced by MPPþ insult
(Supplementary Fig S5C). These findings suggested that
Erk1/2–Elk1 pathway might regulate SOD1 expression to
protect cells against oxidative insult.
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Knocking Down DJ-1 Can Suppress MPPþ- and
Paraquat-Induced Elk1 Phosphorylation and
Erk1/2 Nuclear Translocation
As the protective expression of SOD1 is both DJ-1 and
Erk1/2-Elk1 MAPK dependent, we tried to determine if
there is any relation between DJ-1 and Erk1/2-Elk1. Erk1/
2 distributes throughout the quiescent cells. It is activated
and transferred to the nucleus to phosphorylate its substrates including Elk1 upon the stimulation.31 When the
WT mice were administered MPTP, the protective activation of Elk1 was observed at 60 minutes and 90 minutes,
but this activation was suppressed in the KO mice (see Fig
4D, Supplementary Fig S6). In DJ-1 KD SH-SY5Y cells,
the enhancement of Elk1 phosphorylation induced by
MPPþ or paraquat treatment was also suppressed (Fig 3D
and Supplementary Fig S4B). These results indicated that
DJ-1 is critical for Elk1 activation upon oxidative insult.
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FIGURE 3: SOD1 expression induced by oxidative insults was controlled by Elk1 activation. (A) Bioinformatic analysis showed
that there are 2 potential Elk1 binding sites (red) in the 2kb upstream of the SOD1 transcription initial site. (B) Cross-linking of
protein-DNA in SH-SY5Y cells was performed by treatment with formaldehyde. Proteins together with cross-linked DNA are
immunoprecipitated by rabbit anti-Elk1 and rabbit immunoglobulin G (IgG). The 460bp upstream of the SOD1 transcription initial site was amplified by polymerase chain reaction. GAPDH 5 glyceraldehyde-3-phosphate dehydrogenase; IP 5 immunoprecipitation. (C) SH-SY5Y cells were transfected with Elk1(s383a), the dominant negative form of Elk1. The phosphorylation of
Elk1 and SOD1 expression induced by 1-methyl-4-phenylpyridinium (MPP1) were examined by Western blot. Inactivation of
Elk1 by its dominant negative mutation can suppress the upregulation of SOD1 induced by MPP1. (D) The phosphorylation of
Elk1 and SOD1 expression induced by MPP1 was examined by Western blot in DJ-1 knockdown cells. (E) The activity of SOD1
promoter was measured by luciferase assay. The activation of SOD1 promoter upon MPP1 or paraquat treatment was compared between Elk1(s383a) transfected SH-SY5Y cells and control cells. *p 5 0.007, **p 5 0.024.
DJ-1 is generally thought to function as a molecular chaperone and might act to help the subcellular localization of many proteins.11 The finding that the phosphorylation of Erk1/2 was not affected in the KO mouse
littermate (Fig 4D) led to the prediction that the presence
of DJ-1 might be related to the nuclear translocation of
Erk1/2 instead of its phosphorylation. To elucidate this
possibility, firstly we examined the interaction of Erk2 and
DJ-1. Both coimmunoprecipitation (Co-IP) and immunofluorescence assay all proved the interaction between DJ-1
and Erk1/2 (Supplementary Fig S7). To further screen the
binding region of DJ-1, the protein was divided into 2 truncations (1–100 and 101–189). Co-IP showed that 1 to 100
instead of 101 to 189 truncation was responsible for the
protein binding (Fig 5C). The cysteine residue 106 is critical for the atypical peroxiredoxinlike peroxidase function of
DJ-1,19 and the mutation of this site may affect its activity.
To examine whether the mutation would disrupt the interaction between DJ-1 and Erk1/2, DJ-1 C106A was cotransfected with Erk2 into Hek293t cells; Co-IP showed that
DJ-1 C106A could still bind to Erk2 (Fig 5D).
Erk1/2 can transfer to the nucleus when SH-SY5Y
is insulted by MPPþ, but this nuclear translocation was
October 2011
decreased when DJ-1 was knocked down. The inhibition
of translocation of Erk1/2 in DJ-1 KD cells was confirmed by analyzing the cytoplasm and nuclear fraction
separated from the cells treated with MPPþ for 15, 30,
and 60 minutes (Fig 5A and B). Through immunofluorescence assay, the nuclear translocation of Erk1/2 was
also observed in WT neurons treated with MPPþ for 30
minutes. Overexpression of WT DJ-1 or DJ-1 C106A in
DJ-1 null neurons could restore the nuclear translocation
of Erk1/2 (Fig 5E). These results demonstrated that DJ1 might help the translocation of Erk1/2, sequentially
phosphorylate Elk1, and promote the expression of
SOD1 when cells face oxidative stress.
Reintroduction of DJ-1 to the DJ-1 Null
Neurons Suppressed the Excess Production
of Superoxide Induced by MPPþ Treatment
To confirm the role of DJ-1 in antioxidant protection
and its involved mechanism, WT and DJ-1 KO neurons
were used to examine the nuclear translocation of DJ-1
and ROS production. The results suggested that DJ-1
KO neurons produced more superoxide and peroxide
upon MPPþ treatment compared to the WT neurons
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FIGURE 4: SOD1 expression induced by oxidative insults was dependent on Erk1/2–Elk1 pathway. (A) SH-SY5Y cells were preincubated with 10lM PD98059, the inhibitor of Erk1/2, and dimethylsulfoxide (DMSO) for 2 hours, respectively. The phosphorylation of Erk1/2 and SOD1 expression induced by 1-methyl-4-phenylpyridinium (MPP1) were measured by Western blot. (B)
SH-SY5Y cells were transfected with different amounts of constitutively active MEK-1, and the SOD1 expression was examined.
(C) SH-SY5Y cells were preincubated with PD98059 and DMSO. The activation of SOD1 promoter induced by oxidative insults
was suppressed by Erk1/2 inhibitor. *p 5 0.0335. (D) Wild-type (WT) and DJ-1 knockout mice were administered 1-methyl-4phenyl-1, 2, 3, 6-tetrahydropyrindine (MPTP) for 60 and 90 minutes, and the phosphorylated Erk1/2 and Elk1 levels in substantia nigra were examined by Western blot. The Elk1 activation was inhibited, whereas the phosphorylation of Erk1/2 was not
affected in DJ-1 knockout mice.
(Fig 6 A, B). Reintroduction of WT DJ-1 or DJ-1
C106A into DJ-1 null neurons suppressed the superoxide
production in the DJ-1 null neurons (Fig 6A). WT DJ-1
could also scavenge the excess peroxide in the DJ-1 null
neurons, whereas DJ-1 C106A failed to clear the
increased peroxide (Fig 6B). This suggested that C106
residue of DJ-1 may not be necessary for the regulation
on SOD1 expression, although it is indispensable for the
atypical peroxidase function of DJ-1.
DJ-1 and SOD1 Level Presented Close
Correlation in Salivary Samples of PD Patients
To further verify our finding in PD patients and evaluate
its potential clinical application, the salivary samples
from PD patients of different stages and controls (Supplementary Table) were collected according to the
method reported by Devic et al.32 The protein levels of
DJ-1 and SOD1 were examined by Western blot. Preliminary study showed a close correlation between the two
proteins (Supplementary Fig S8), confirming that DJ-1
may participate in the regulation of SOD1 expression.
Discussion
Oxidative stress is the state of imbalance between ROS
production and its clearance. O
2 , a major ROS, is
increased in inefficient energy metabolism, hypoxia, reper596
fusion, and environmental toxins exposure.33 Cells have endogenous antioxidant proteins such as SODs to process
this radical and reduce its toxicity.33 SODs are the important antioxidant factors in the cells and may play an important role in the pathogenesis of PD.34 SOD1 transgenic
mice are resistant to MPTP-induced neurotoxicity, and
overexpressing SOD1 enhances the survival of transplanted
neurons in the PD rat model.35,36 In a drosophila PD
model, overexpression of SOD1 can protect against the dopaminergic neuronal loss induced by mutant a-synuclein.37
Our in vivo and in vitro studies also showed that SOD1
expression is induced when WT mice or cells are attacked
by oxidative stress. The study of DJ-1 KD cells also found
failure of SOD1 upregulation, increased ROS production
and cell death upon oxidative stimulation. Interestingly, the
cell death in DJ-1 KD cells treated with MPPþ or paraquat is decreased if SOD1 is reintroduced. These data provide additional evidence that links DJ-1 with antioxidant
stress, suggesting that DJ-1 may protect cells from oxidative damage through increasing SOD1 expression. SODs
are mainly composed of SOD1, which is principally present in cytosolic and SOD2, which is localized in mitochondria. Zhong and Xu reported that DJ-1 could increase
SOD2 expression.38 In the results of our experiments, only
SOD1 expression is affected by DJ-1 deficiency; there is
no significant difference in SOD2 level between WT and
DJ-1 null mice. This indicated that DJ-1 deficiency may
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FIGURE 5: The interaction of DJ-1 and ERK1/2 helped the nuclear translocation of ERK1/2. (A) A representative fluorescent
microscope image shows the nuclear translocation of Erk1/2 upon MPP1 insult (d); this translocation is inhibited when DJ-1
expression is downregulated (j). (B) The scramble and DJ-1 knockdown SH-SY5Y cells were treated with 1-methyl-4-phenylpyridinium (MPP1) for the indicated times. The cytoplasm and nuclear fractions were separated, and the nuclear and cytosol Erk1/
2 were assayed by Western blot. The results indicated the decreased nuclear translocation of Erk1/2 in DJ-1 knockdown cells.
(C) The 1 to 110 or 110 to 189 truncation domain of DJ-1 was cotransfected with Flag-Erk2 to the Hek293t cells. Immunoprecipitation assay showed that the 1 to 110 domain of DJ-1 was responsible for its interaction with Flag-Erk2. (D) Coimmunoprecipitation (Co-IP) assay showed that DJ-1 C106A still could bind to Erk1/2. WCL = whole cell lysates, IB = Immunoblotting. (E)
Wild-type (wt) or DJ-1 knockout (KO) neurons were treated with MPP1 for 30 minutes. The result showed that the nuclear
translocation of Erk1/2 induced by MPP1 was suppressed in the DJ-1 null neurons (e). Reintroduction of DJ-1 and DJ-1 C106A
could restore the nuclear translocation of Erk1/2 in DJ-1 KO neurons (j, m).
only disrupt the expression of SOD1 upon oxidative stress
instead of inhibiting both SOD1 and SOD2 expression.
Using mobility shift assay and site mutagenesis,
Chang MS et al identified Elk1 as a positive regulator in
the SOD1 promoter.28 Our study confirmed that Elk1
could bind to the SOD1 promoter region and enhance
SOD1 expression. The mutation of the phosphorylation
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site of Elk1 will cause decreased SOD1 expression and
increased ROS production upon oxidative attack.
A number of studies have suggested that DJ-1
could function as a molecular chaperon and will redistribute to the nucleus in cells treated with oxidative
insult. Our in vivo and in vitro data revealed that DJ-1
could interact with Erk1/2 and mediate its nuclear
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FIGURE 6: Reintroduce of DJ-1 to the DJ-1 null neurons
suppressed the excess production of superoxide induced by
1-methyl-4-phenylpyridinium (MPP1) treatment. (A) The
wild-type (WT) primary neurons or the DJ-1 null neurons
transfected with the indicated constructs for 48 hours were
treated with 100lM MPP1 for 24 hours, and the superoxide
level was measured by dihydroethidium (DHE) probe. The
results showed that MPP1 treatment could produce more
superoxide in the DJ-1 knockout (KO) neurons. Both DJ-1
and DJ-1 C106A could suppress the excessive production of
superoxide in DJ-1 KO neurons. (B) The WT primary neurons
or the DJ-1 null neurons transfected with the indicated constructs for 48 hours were treated with 100lM MPP1 for 24
hours, and the hydrogen peroxide level was measured by
Amplex Red probe. The results showed that DJ-1 instead of
DJ-1 C106A could suppress the excessive production of peroxide in DJ-1 KO neurons. * p < 0.05, ** p < 0.01.
translocation to counteract oxidative stimulation. Loss of
DJ-1 will affect the redistribution of Erk1/2, sequentially
decrease the activation of Elk1 and the expression of
SOD1, and finally weaken the antioxidant response of
cells and increase their susceptibility to oxidative stress.
A previous publication has reported that DJ-1 is an
atypical peroxiredoxinlike peroxidase that scavenges
H2O2.19 Our experiment showed that DJ-1 C106A mutation did not affect the binding between DJ-1 and Erk1/2.
Reintroduction of DJ-1 mutant C106A into DJ-1 null
neurons treated with MPPþ could restore the nuclear
translocation of Erk1/2 and reduce the superoxide level of
the cells. It seems that DJ-1 C106A mutation did not
affect the upregulation of DJ-1 on SOD1 expression upon
oxidative insult. Cysteine residue 106 is critical for the
function of DJ-1 as an atypical peroxiredoxinlike peroxidase. The mutation of this residue will inactivate its ability
to react with peroxides. The peroxide level in DJ-1 null
neurons transfected with DJ-1 C106A is even higher than
that of DJ-1 null neurons. This suggests that DJ-1 C106A
mutation impaired the function of DJ-1 as an atypical
peroxiredoxinlike peroxidase and could not scavenge the
peroxides produced by SOD1. These results indicate that
increased superoxide and hydrogen peroxide in DJ-1 KD
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cells or DJ-1 KO neurons are not the indirect consequence of the effects of DJ-1 on peroxide levels. It also
indicates that under oxidative insult, Cys-106 oxidation
only is not capable of protecting neurons from oxidative
stress. The upregulation of SOD1 induced by DJ-1 and
the following catalase or peroxidase enhancement may be
needed at this time to help clean the excessive ROS.
Plasma membrane nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can transfer electrons
from NADPH to molecular oxygen to produce superoxide. It is thought that NADPH oxidase is the enzyme primarily responsible for generating extramitochondrial or cytosolic ROS. MPPþ is generally thought to increase ROS
production through its effects on mitochondria. However,
there are several reports showing that knocking down
NADPH oxidase subunit or inhibiting NADPH oxidase
blocked MPPþ-caused cell death.39–41 This suggests that
MPPþ treatment induces cytosolic ROS, including superoxide as well as mitochondrial ROS. Scavenging the excessive cytosolic superoxide through upregulated SOD1 may
help cells antagonize the oxidant insult. Our experiments
showed the increase of cytosolic superoxide and mitochondrial peroxide in DJ-1 KD cells, indicating that DJ-1 deficiency would impair the activity of peroxiredoxinlike peroxidase and upregulation of SOD1 as well as during
oxidative stress. They also demonstrated that cytosolic
superoxide was an important resource of ROS as well in
DJ-1 KD cells treated with MPPþ.
It was reported that human DJ-1 Arabidopsis thaliana homologue DJ-1a mediated cytosolic SOD1 activation in a post-translational manner,42 and that loss-offunction AtDJ-1a would cause cell death. These results
and ours strongly suggest that the cytosolic antioxidant
response plays the same important role as mitochondria
in the antioxidant defense system of cells, and that DJ-1
conferred oxidative stress protection through its regulation of SOD1 activity or expression. The different mechanism involved in the regulation of DJ-1 on SOD1 in 2
reports may be caused by the different model.
Oxidative stress plays an important role in the
pathogenesis of PD. DJ-1 is an important antioxidant
factor, and salivary DJ-1 may be considered the biomarker of PD. The increased salivary DJ-1 in early stage
PD patients was similar to the findings of Dr Jin Zhang’s
laboratory at the University of Washington.32 Dr Zhang
communicated that most of their samples were from relative early stage PD patients. This suggests that cells may
try to upregulate DJ-1 to antagonize oxidative insult in
the early stage of PD. The close correlation between DJ-1
and SOD1 levels further defined the role of DJ-1 in the
regulation of SOD1 expression. However, these are only
preliminary data based on a limited number of patient
Volume 70, No. 4
Wang et al: DJ-1 Modulates SOD1 Activity
samples. The further study of a large sample size is needed
to confirm the correlation between DJ-1 and SOD1 and
to validate salivary DJ-1 as a diagnostic marker of PD.
Our studies elucidate a new molecular mechanism involved
in the antioxidant function of DJ-1 and provide important
insight into the pathogenesis of PD.
Acknowledgment
This work was supported by the National Program of
Basic Research (2007CB947900, J.D. 2010CB945200,
2011CB504104; S.C.) of China, Natural Science Fund
(30700888, C.L. 30770732, J.D. 30872729 and
30971031; S.C. 81028007, J.Z., 30828031, J.Z.), Key
Discipline Program of Shanghai Municipality (S30202;
S.C.), Shanghai Key Project of Basic Science Research
(10411954500 S.C.), and Program for Outstanding
Medical Academic Leader of Shanghai (LJ 06003, S.C.).
We thank Dr A. D. Sharrocks, Dr R. T. Hay, and
Dr. R. Seger for providing us with Elk1, dominant negative Elk1, and constitutively active MEK1 constructs, respectively; and Dr J. Shen for providing the DJ-1 knockout mice.
Potential Conflicts of Interest
Nothing to report.
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